ProjectSummary&Abstract Obligateintracellularparasites,whichincludevirusesaswellascertainbacteriaandeukaryotes,requireahost cellforgrowth.Theyalsohavetheuniquephenomenaofundergoingreductivegenomeevolutionasaresultof theirparasiticlifestyle,stealingmanyessentialnutrientsandmetabolitesfromtheirhost.Asaconsequence, thesepathogensnolongercontainthegenesandpathwaystosynthesizetheirownnutrientsormetabolites, revealinganabsolutedependenceandvulnerabilityoftheirexistenceonhostsystems.Gram-negative bacteriaoftheorderRickettsialesarecomprisedofadiversegroupoforganismsthatallshareacommon obligateintracellularlifestyleandareassociatedwithhumandiseasessuchasepidemictyphusandRocky Mountainspottedfever.WediscoveredthatmembersoftheRickettsiageneralackthebiosyntheticpathwayto produceessentialisoprenoidprecursorsthatareusedtomakeacellwallandcomponentsrequiredfor oxidativephosphorylation.Yet,theymaintainenzymesforisoprenoidconversionandutilization,suggesting theyscavengeisoprenoidprecursorsdirectlyfromthehost.Theessentialityofisoprenoidbiosynthesisto bacterialsurvival,coupledwiththepredicteddependenceoftheseobligatepathogensonhostisoprenoids, suggestsanexploitableweaknessofthesemetabolicparasites.MypreliminarystudiessuggestthatRickettsia specieshaverewiredtheirisoprenoidbiosyntheticpathwaysinordertoscavengehostisoprenoids.HereI proposeaseriesofexperimentstodetailthisgeneticrewiringofanessentialbiosyntheticpathwayandto determinewhetherthismetabolicdependencyexposesanunexploredtargetforantibiotictherapeutics.First,I willcharacterizethemorphologicalandphysiologicaleffectsofisoprenoiddepletioninAim1.Then,to implicatethehostcellasthesourceofbacterialisoprenoids,inAim2,Iwillinhibitthehostisoprenoid biosyntheticpathwaywithstatinsanddeterminetheeffectofRickettsialgrowthwithwildtypeR.parkeri comparedtoamutantimpairedinisoprenoidutilization.Then,toidentifybacterialcomponentsthatmediate isoprenoidscavengingfromthehost,inAim3,Iwillperformtargetedgenedisruptionsofputativeisoprenoid transporterstodeterminetheeffectonisoprenoidimportintothebacterialcellandadditionally,Iwilltakean unbiasedapproachtoidentifyadditionalfactorsthatmediateisoprenoidutilizationandtransport.Theultimate goalofthisprojectistodetailthegeneticrewiringofapathogen'smetabolicpathway.However,the conclusionsandtechniquesdevelopedfromthisstudywillbecriticalinthediscoveryofspecies-specific chemicaltherapeuticsthatinterruptmetabolicparasitismbyapathogen.Theresultsofthisstudywillopen avenuestonewmethodsofantibioticdrugdiscoveryfordiseasesofobligateintracellularpathogensincluding Plasmodium,Chlamydia,andToxoplasma.